Cancer is caused by a series of genetic alterations destroying the normal mechanisms that control the cell cycle, differentiation, and morphology. A large number of natural compounds have been isolated based on their ability to restore cells having abnormal morphology, to induce cell differentiation, and to stop uncontrolled cell cycles in various cancer cells and transformed cells. Trichostatin A (TSA) (Sugita K et al. (1992). Cancer Res., 52, 168-172) and trapoxin (Itazaki H et al. (1990). J. Antibiot., 43, 1524-1532) have been isolated as substances capable of suppressing transformation. These substances also induce cell differentiation and cell cycle arrest. However, it was not clear how these materials showed such tumor-suppressing activity.
It is the current belief that histone deacetylases (HDAC) are the target of these drugs. Actually, TSA and trapoxin inhibit HDAC activity at the same concentration in which they show antitumor activity (Yoshida et al., 1990, J. Biol. Chem., 265:17174-17179; Kijima M et al., 1993 J. Biol. Chem., 268, 22429-22435). Rapidly accumulating findings suggest that acetylation and deacetylation of histone and non-histone proteins play an important role in the transcriptional control of eukaryotic cells (Wolffe A P and Pruss D. (1996). Cell, 84, 817-819; Wade P A et al. (1997). TIBS, 22, 128-132; Pazin M J and Kadonaga J T. (1997). Cell, 89, 325-328; Struhl K. (1998). Genes Dev., 12, 599-606; Kuo M H and Allis C D. (1998). Bioessays, 20, 615-626). Since many transcription factors, transcription coactivators, and basic transcription initiation complex proteins have histone acetyltransferase activity, it became clear that acetylation of histone plays an important role in the initiation and promotion of transcription. Furthermore, the recent cloning of some of the HDACs, and the fact transcriptional repressors, and transcription corepressors form complexes with HDAC, gradually revealed that HDAC plays an important role in transcriptional repression (Wolffe A P. (1997). Nature, 387, 16-17). As HDAC inhibitors show antitumor activity, HDAC may repress transcription of a group of antitumor genes whose products induce cell proliferation arrest or cell differentiation (DePinho R A. (1998). Nature, 391, 533-536).
The present inventors earlier proved that sodium butyrate, which is well known as a differentiation inducer and acts as a HDAC inhibitor at micro molar concentrations, induced the expression of p21/WAF1/Cip1, which is a cyclin-CDK inhibitor (a negative regulator of cell cycle) acting independently of p53 (Nakano K et al. (1997). J. Biol. Chem., 272, 22199-22206). They also reported that both sodium butyrate and TSA activated p21/WAF1/Cip1 gene promoter through the Sp1 binding sequence (Sowa Y et al. (1997). Biochem. Biophys. Res. Comm., 241, 142-150). Interestingly, it was reported recently that the Sp1 binding site in p21/WAF1/Cip1 promoter was also involved in the induction of p21/WAF1/Cip1 by TGF-β, phorbol ester, okadaic acid, progesterone, or geranylgeranyl-transferase I inhibitor GGTI-298 (Datto M B et al. (1995). J. Biol. Chem., 270, 28623-28628; Biggs J R et al. (1996). J. Biol. Chem., 271, 901-906; Adnane J et al. (1998). Mol. Cell. Biol., 18, 6962-6970; Owen G I et al. (1998). J. Biol. Chem., 273, 10696-10701). Among these, TGF-β and GGTI-298 have been reported to induce transcription of p21/WAF1/Cip1 by enhancing the transcriptional activity of Sp1, whereas progesterone accomplishes the same through Sp1 and CBP/p300 (Li J M et al. (1998). Nucleic Acids Res., 26, 2449-2456; Owen G I et al. (1998). J. Biol. Chem., 273, 10696-10701).
These reports are thought to suggest the induction of transcription by SP1-mediated activation of histone acetyltransferase. Histone acetylation is thought to be involved in the transcriptional activation of many genes. In contrast, histone deacetylation is considered to be involved in transcriptional repression, although the detailed mechanism is not clear. Recently, it was reported that transcriptional repressors N-CoR and SMRT repressed the transcription specific to a DNA sequence by binding to intranuclear transcription factors (Horlein A J et al. (1995). Nature, 377, 397-404; Kurokawa R et al. (1995). Nature, 377, 451-454; Chen J D and Evans R M. (1995). Nature, 377, 454-457). As these factors bind to HDAC simultaneously and form a higher order complex, it is suggested that the rigid organization of chromatin, which is mediated by histone deacetylation, represses transcription (Pazin M J and Kadonaga J T. (1997). Cell, 89, 325-328; Heinzel T et al. (1997). Nature, 387, 43-48; Alland L et al. (1997) Nature, 387, 49-55). In fact, studies using a fusion protein of promyelocytic leukemia or promyelocytic leukemia zinc-finger protein and retinoic acid receptor revealed that the binding of HDAC was necessary for transcriptional repression (Lin R J et al. (1998). Nature, 391, 811-814; Grignani F et al. (1998). Nature, 391, 815-818; He L Z et al. (1998). Nature Genet., 18, 126-135). A similar HDAC-mediated mechanism of transcriptional repression specific to a DNA sequence was revealed also in the case of Myc/Mad/Max (Hassig C et al. (1997). Cell, 89, 341-347; Laherty C D et al. (1997). Cell, 89, 349-356), E2F/Rb (Brehm A et al. (1998). Nature, 391, 597-601; Magnaghi-Jaulin L et al. (1998). Nature, 391, 601-605; Luo R X et al. (1998). Cell, 92, 463-473), and in the case of DNA methylation (Nan X et al. (1998). Nature, 393, 386-389; Jones P L et al. (1998). Nature Genet., 19, 187-191). However, it was not clear whether or not a specific transcription factor capable of binding to the Sp1 binding sequence mediates the transcriptional activation signal by a HDAC inhibitor.